Fimbrin associated with Pmk1 to regulate the actin assembly during Magnaporthe oryzae hyphal growth and infection

The dynamic assembly of the actin cytoskeleton is vital for Magnaporthe oryzae development and host infection. The actin-related protein MoFim1 is a key factor for organizing the M. oryzae actin cytoskeleton. Currently, how MoFim1 is regulated in M. oryzae to precisely rearrange the actin cytoskeleton is unclear. In this study, we found that MoFim1 associates with the M. oryzae mitogen-activated protein (MAP) kinase Pmk1 to regulate actin assembly. MoFim1 directly interacted with Pmk1, and the phosphorylation level of MoFim1 was decreased in Δpmk1, which led to a change in the subcellular distribution of MoFim1 in the hyphae of Δpmk1. Moreover, the actin cytoskeleton was aberrantly organized at the hyphal tip in the Δpmk1, which was similar to what was observed in the Δmofim1 during hyphal growth. Furthermore, phosphorylation analysis revealed that Pmk1 could phosphorylate MoFim1 at serine 94. Loss of phosphorylation of MoFim1 at serine 94 decreased actin bundling activity. Additionally, the expression of the site mutant of MoFim1 S94D (in which serine 94 was replaced with aspartate to mimic phosphorylation) in Δpmk1 could reverse the defects in actin organization and hyphal growth in Δpmk1. It also partially rescues the formation of appressorium failure in Δpmk1. Taken together, these findings suggest a regulatory mechanism in which Pmk1 phosphorylates MoFim1 to regulate the assembly of the actin cytoskeleton during hyphal development and pathogenesis. Supplementary Information The online version contains supplementary material available at 10.1007/s44154-023-00147-5.


Introduction
Rice blast caused by the fungus Magnaporthe oryzae is one of the most destructive diseases of rice and is a main crop nourishing more than half of the world's population.A great amount of harvest loss occurs in all rice-growing regions of the world every year (Wilson and Talbot 2009).The basic unit of growth of M. oryzae is the hypha, which consists of tube-like structures with a hemispherical or hemiellipsoidal apical region (Fernandez and Orth 2018).The polar growth of M. oryzae hyphae is a fundamental property for cell development and is accompanied by the transport of cellular components to specific sites in the cell.It requires the continuous synthesis and selective targeting of proteins, lipids, and cell wall materials to specific domains at the apex of the hyphal plasma membrane (Riquelme 2013).During M. oryzae infection, the polar growth of hypha occurring at the hyphal tip is associated with endocytosis and effector secretion, which is critical for the invasive hypha extending across the plasmodesmata to spread in the host plant cell (Wendland and Walther 2006).Thus, understanding the biology of fungal cells is required for revealing the infection mechanism of M. oryzae.
Mitogen-activated protein (MAP) kinase cascades play important roles in environmental signal sensing, development and host infection in plant fungal pathogens.The MAPK signaling pathway is important for plant infection in more than 20 plant pathogenic fungi (Li et al. 2012).It has been revealed that in M. oryzae, a mitogen-activated protein kinase (MST11-MST7-Pmk1) MAP kinase cascade exists that guides infection-related morphogenesis (Zhao et al. 2005).A MAPK docking site in Mst7 is essential for Pmk1 activation during M. oryzae infection (Zhao and Xu 2007).Mst50 functions as an adapter protein of the Mst11-Mst7-Pmk1 cascade that is essential for appressorium formation (Park et al. 2006;Li et al. 2017a).In the model organism Saccharomyces cerevisiae, there are five MAPK proteins (Fus3, Kss1, Slt2, Hog1, and Smk1) (Waltermann and Klipp 2010;Xu and Hamer 1996).However, in most plant pathogenic fungi, only three MAPK genes orthologous to Hog1, Fus3/Kss1, and Slt2 (Jiang et al. 2018) are present.Pmk1 is an orthologue of Fus/Kss1 and was suggested to be critical for fungal development and appressorium formation in appressorium-forming fungi, including the rice blast fungus M. oryzae, Colletotrichum lagenarium, and Colletotrichum gloeosporioides (Xu and Hamer 1996;Takano et al. 2000;He et al. 2017;Abah et al. 2023).Mutants of PMK1 in M. oryzae fail to construct specialized infection structures known as appressorium and fail to develop invasive hyphae in rice plants (Xu and Hamer 1996).Detection of green fluorescence protein (GFP)-labelled Pmk1 showed that Pmk1 was expressed in vegetative hyphae, conidia, and germ tubes and that the expression of GFP-Pmk1 increased in appressorium and developing conidia.Especially in the appressorium, GFP-Pmk1 fluorescence can be observed in the nucleus (Bruno et al. 2004), indicating the roles of Pmk1 in these processes.The M. oryzae PMK1 mutant could not develop a mature appressorium from conidial germlings on a hydrophobic surface (Xu and Hamer 1996).Lipid and glycogen mobilization and autophagy were disrupted in the PMK1 mutant (Thines et al. 2000;Veneault-Fourrey et al. 2006).The PMK1 mutant hyphae could not penetrate the neighboring host cell and thus failed to undergo cell-to-cell movement (Sakulkoo et al. 2018).Recently, Pmk1 was shown to trigger the infection process through phosphorylation of the Hox7 homeobox transcription factor, which is associated with the development of M. oryzae appressorium, including cell division, autophagy, turgor generation and melanin biosynthesis (Oses-Ruiz et al. 2021).These findings indicated that Pmk1 is particularly important for M. oryzae infection, and the complex molecular mechanism underlying Pmk1 infection has become a hot topic in M. oryzae research.
The actin cytoskeleton plays a central role in cell morphogenesis and maintaining hyphal polar growth in filamentous fungi (Berepiki et al. 2011).Three high-order filamentous actin (F-actin) structures with distinct functions, actin rings, patches, and cables, were found to be organized in M. oryzae.The actin rings are suggested to associate with the formation of a septum, and the actin patches localize to subapical regions and are involved in endocytosis.Actin cables are linear bundles of short actin filaments that are present at the apexes of hyphae (Riquelme 2013).At the hyphal tip of M. oryzae, the actin cytoskeleton is dynamically assembled as a spatial structure that guides the growth of the cell (Li et al. 2020).The assembly of actin is guided by numerous actin-related proteins, such as motor proteins, actin bundle proteins, and septins.Mutants of these actin-related protein genes in M. oryzae exhibit severe defects in either development or host infection (Guo et al. 2017;Motaung and Tsilo 2017;Tang et al. 2018;Xu et al. 2021).Fimbrin is a typical actin bundle protein, and M. oryzae fimbrin are known to localize to the hyphal tip region (Gupta et al. 2015) and to regulate actin assembly at the hyphal tip during polar growth.Knockout of fimbrin (MoFim1) in M. oryzae could abolish the infection ability (Li et al. 2020).Although we know the importance of MoFim1 in M. oryzae development and pathogenesis, we do not know how it is regulated.
In this study, we found that M. oryzae MoFim1 could directly interact with Pmk1 and that Pmk1 could affect the phosphorylation level of MoFim1.Phosphorylation of MoFim1 was decreased in the Δpmk1 strain.Dephosphorylation of MoFim1 resulted in altered localization and decreased actin bundling activity, thus affecting the organization of the actin cytoskeleton in hyphae.We provide evidence that Pmk1 could regulate the actin cytoskeleton through modulation of the phosphorylation of the actin-related protein MoFim1 during M. oryzae development and pathogenesis.

MoFim1 interacts with Pmk1
Previously, we found that the M. oryzae fimbrin protein MoFim1 is an actin-binding protein that is required for the endocytosis, growth, development, and full virulence of M. oryzae (Li et al. 2020).To further explore the mechanism by which MoFim1 functions, we employed protein immunoprecipitation mass spectrometry (IP-MS) to identify putative MoFim1-interacting proteins.By expressing the MoFim1-GFP and GFP constructs and using GFP beads to isolate MoFim1-GFP-interacting proteins followed by mass spectrometry (MS) analysis, we identified six proteins that may interact with MoFim1 including the actin motor protein Myosin 2, the Arp2/3 complex subunit protein, Septin 3, Pmk1, the Woronin body protein (Hex1) and a dynamin protein (Fig. 1A).As Pmk1 is vital to M. oryzae (Xu and Hamer 1996;Sakulkoo et al. 2018), we next verified the interaction between Pmk1 and MoFim1.We employed the yeast two-hybrid assay (Y2H) to evaluate the interactions.Yeast cells expressing the AD-MoFim1 and BD-PMK1 constructs were grown on SD/-Leu/-Trp DO (DDO) plates and SD/-Leu/-Trp/-Ade/-His DO (QDO) plates (Fig. 1B).The results showed that Pmk1 and MoFim1 could interact in this system.MoFim1 contains three motifs, namely, the N-terminal EF-hand (EF) motif and two actin-binding domains (ABD1 and ABD2), in tandem (Fig. 1C).We further found that Pmk1 could interact with the ABD1 motif (Fig. 1D).We next used a co-IP experiment to verify the interaction of the two proteins.We then expressed MoFim1-GFP and Pmk1-mCherry driven by their native promoters in M. oryzae via co-IP (Supplemental Fig. 1).The results showed that Pmk1 coimmunoprecipitated with MoFim1 (Fig. 1E).In addition, bimolecular fluorescence complementation (BiFC) assays further substantiated the interaction between MoFim1 and Pmk1 (Fig. 1F).We noticed that the interactions took place at the hyphal tip area.We also investigated the subcellular distribution of Pmk1.The results showed that Pmk1 was expressed in M. oryzae hyphae, the germ tube and the appressorium (Supplemental Fig. 2).Taken together, these results indicate that MoFim1 can interact with Pmk1.

Phosphorylation of MoFim1 was decreased in the Δpmk1
A mutant of MoFim1 was previously generated in our laboratory (Li et al. 2020).We then generated mutant of PMK1 in M. oryzae to further study their relationships.The generation of a PMK1 knockout mutant in M. oryzae was performed (Supplemental Fig. 3).Next, we tested the infection ability of Δpmk1 and Δmofim1, which we previously obtained (Li et al. 2020).We infected the rice leaves in the same region on the plate for the SRB-grown cultures of the WT, Δpmk1, and Δmofim1 and their complemented strains.The results showed that the deletion of PMK1 completely abolished the infection ability, as previously reported (Xu and Hamer 1996;Sakulkoo et al. 2018), and that the Δmofim1 mostly lost its ability to infect.The complemented strains could rescue their infections (Fig. 2A and B).
We next expressed MoFim1-GFP driven by its native promoter in the WT and Δpmk1 strains.Total proteins from hyphae were examined and incubated with anti-GFP beads.Then, Western blot analysis was performed using both a GFP antibody and a phosphorylation antibody.The results indicated that MoFim1 could be phosphorylated.In addition, phosphorylation of MoFim1 was decreased in the Δpmk1 compared with the WT (Fig. 2C).We next added lambda protein phosphatase (λ-PP) to test its phosphorylation.The results showed that phosphorylation of MoFim1 decreased markedly, indicating confidence in the phosphorylation (Fig. 2D).We also analyzed the phosphorylation of MoFim1 in the appressorium.Δpmk1 could not form an appressorium (Fig. 2E).Moreover, phosphorylation of MoFim1 in the pmk1 appressorium significantly decreased compared with that in the WT (Fig. 2F).We further analyzed the phosphorylation of the proteins in both the WT and the Δmofim1 by mass spectrometry.The results showed that the phosphorylation of MoFim1 at Ser94 and Ser117 was markedly lower in the Δpmk1 strain than in the WT strain (Fig. 2G).

Pmk1 phosphorylated MoFim1 at Ser94, which is critical for its actin bundling activity
To verify that Pmk1 phosphorylates MoFim1, we expressed and purified His-tagged Pmk1 and MoFim1 proteins, which included S94A (with the MoFim1 94 serine changed to alanine to mimic dephosphorylation), S94D (with the MoFim1 94 serine changed to aspartic acid to mimic phosphorylation) and S117A (with the MoFim1 117 serine changed to alanine to mimic dephosphorylation), in E. coli BL21 (DE3) cells.Pmk1 alone or incubated with MoFim1 was analysed using a phosphorylation antibody.The results showed that Pmk1 could autophosphorylate (Fig. 3A, lane 1).In the presence of Pmk1, MoFim1 was phosphorylated.Along with the increasing amount of Pmk1, the phosphorylation of MoFim1 increased (Fig. 3A, lanes 3-5).Next, we incubated Pmk1 with S94A and S117A, and the results showed that S94A but not S117A lost phosphorylation (Fig. 3B, lanes 3 and 4).These results indicated that Pmk1 could regulate the phosphorylation of MoFim1 at serine 94.
As the main function of MoFim1 is to bundle the actin cytoskeleton, we next investigated whether phosphorylation of MoFim1 affects its actin bundling activity.We then incubated filamentous actin (F-actin) with these purified MoFim1 or Pmk1 proteins.The results showed that F-actin exhibited thin filaments (Fig. 3C), and incubation with Pmk1 did not affect its polymerization.When MoFim1 was added to F-actin, actin bundles appeared.Furthermore, the presence of Pmk1 or S94D induced thicker actin bundles than did the presence of MoFim1 alone.However, S94A exhibited weaker actin bundle activity than MoFim1.The statistical analysis further supported these conclusions (Fig. 3D).Taken together, these results showed that phosphorylation of MoFim1 at serine 94 is critical for its actin bundling activity.

Localization of MoFim1 was changed in the Δpmk1
The above results showed that the phosphorylation of MoFim1 was compromised when PMK1 was knocked out.We then checked the cellular localization of MoFim1 in both the WT and Δpmk1.Time-lapse imaging of vegetative hyphae revealed that MoFim1-GFP formed dense patches in the cytoplasm, the collar in the subapical region and the Spitzenkörper (Spk) region at the hyphal tip.(Fig. 4A, Supplemental Movie 1).We then investigated distribution of MoFim1 in Δpmk1.We found that, in the hyphal subapical region, the localization of MoFim1 did not change significantly.However, in the hyphal tip, the distribution of MoFim1 was abolished (Fig. 4B, Supplemental Movie 2).In the enlarged regions of the hyphal tip, the distribution of MoFim1-GFP in the Δpmk1 was lost compared with that in the WT (Fig. 4C  and D).The line scan analysis further supported these findings (Fig. 4E, F and G).We also found that the hyphal growth rate decreased in the Δpmk1 (Fig. 4H).Taken together, these results indicated that Pmk1 is required for the hyphal tip localization of MoFim1.Fimbrin is an actin-related protein that can facilitate the formation of actin bundles.We then investigated actin organization in both Δpmk1 and Δmofim1.We expressed the actin-labelling peptide Lifeact in combination with GFP in M. oryzae strains.Through live-cell imaging, we found that actin was dynamically assembled at the hyphal tip in the hyphae of the WT.We observed that actin accumulated at the hyphal tip where Spk was located, and some actin cables connected to Spk (Fig. 5A and C, Supplemental Movies 3 and 5).However, in both Δpmk1 and Δmofim1, we found that there was no stable actin accumulation in the Spk region (Fig. 5B and D, Supplemental Movies 4 and 6).In addition, when S94D was expressed in Δpmk1, actin accumulation at Spk was partially restored (Fig. 5E, Supplemental Movie 7).The enlarged regions of the hyphal tips of these M. oryzae strains further showed that actin was distributed at Spk (Fig. 5F-J).

S94D expression could restore the hyphal growth of Δpmk1
As MoFim1 is required for the hyphal growth of M. oryzae, we then observed the growth of the Δpmk1, Δmofim1 and their complementary strains.We cultured these M. oryzae strains on straw rice bran (SRB) medium.The results showed that the Δmofim1 exhibited a slower growth rate than the WT (Fig. 6).Additionally, expression of MoFim1 and S94D driven by the native promoter of MoFim1 in Δmofim1 rescued the phenotype (Fig. 6).The growth of the Δpmk1 also decreased compared with that of the WT and the complemented strain.We also expressed MoFim1s, including MoFim1, S94A and S94D, in Δpmk1.We found that the expression of MoFim1 and S94A could not reverse the growth-inhibiting phenotype, but S94D could increase the growth of Δpmk1.The statistical analysis further supported these conclusions (Fig. 6B).

S94D partially rescues Δpmk1-induced appressorium formation failure
We next studied whether S94D could rescue the infection defects of Δpmk1.We collected actin-labelled conidia from the WT, Δpmk1, and Δpmk1 strains expressing S94D-mCherry and S94A-mCherry.After induction on hydrophobic glass for 2 h, these conidia developed an elongated germ tube.With respect to the WT, a dense actin network accumulated at the tip of the germ tube (Fig. 7A, middle panel).However, in the Δpmk1, the fluorescence in the actin network was much weaker in the tip region of the germ tube than in the WT (Fig. 7B, middle panel).The expression of S94D-mCherry but not S94A-mCherry in the Δpmk1 strain recovered dense actin accumulation at the germ tube tip (Fig. 7C and D).Furthermore, after 8 h of induction, the WT successfully developed an appressorium, and an actin ring appeared in the cell (Fig. 7A, right panel).Compared with the WT, the Δpmk1 could not form an appressorium, and no actin rings were observed (Fig. 7B, right panel).Interestingly, in the Δpmk1 strain that expressed S94D-mCherry but not S94A-mCherry, the tip of the germ tube usually expanded to form a smaller appressorium.Additionally, the fluorescence of S94D-mCherry and actin was distributed at the periphery of these cells (Fig. 7C).These results indicated that S94D contributed to the development of an appressorium for Δpmk1.We next assessed whether this appressorium could penetrate and infect rice cells.These conidia were used to infect the rice sheath cells.After 12 h of infection, the WT M. oryzae penetrated the rice sheath cells (Fig. 7E, left panel).However, the Δpmk1 expressing S94D-mCherry could not penetrate into the rice cells even though it formed an appressorium-like structure (Fig. 7E, right panel).We then calculated the appressorium formation rate and found that the expression of S94D-mCherry could induce 15% of the germ tubes to develop an appressorium-like structure in Δpmk1 (Fig. 7F and G).And we further observed that in the WT and the S94D complemented Δmofim1 stain, it formed an actin ring in the appressorium cell.But in the S94A complemented Δmofim1 stain, it can not develop an intact actin ring (Fig. 7H).Taken together, these results indicated that the expression of S94D in Δpmk1 could partially rescue the morphological defects of Δpmk1 by inducing an appressorium but could not rescue the penetration defects associated with infection of host cells.

Discussion
Rice blast disease is one of the greatest threats to global food security (Talbot 2003).The underlying biological processes of development and plant infection, including the regulation of hyphal growth, appressorium formation and invasive hyphal proliferation in plant cells, are critical for understanding the mechanism of M. oryzae pathogenesis.The actin cytoskeleton is a highly dynamic system that can organize distinct spatial structures in different cells of M. oryzae.They could assemble as actin patches and actin bundles in the growing hyphae (Li et al. 2020), actin ring in the formation of septum (Delgado-Alvarez et al. 2010) and hetero-oligomeric ring like structure in the infection cell, the appressorium to rupture the plant cell wall (Dagdas et al. 2012).Investigating the regulation of actin assembly is helpful for the development of new fungicides for fungal disease control (He et al. 2020).In the present study, we found that the actin-related protein MoFim1 may be regulated by Pmk1 through phosphorylation.This may represent a new regulation of fimbrin mediated actin organization pattern.
Fimbrin is a major regulator of actin organization, is conserved among eukaryotes and plays important roles in a variety of cellular processes (Skau et al. 2011).Fimbrin can be phosphorylated by a kinase.For example, budding yeast fimbrin can be phosphorylated by cyclindependent kinase 1 (Cdk1) at threonine 103 to stabilize the N-terminal domain of fimbrin and modulate F-actin binding to regulate actin cable assembly (Miao et al. 2016).In M. oryzae, fimbrin localizes to the hyphal tip region (Gupta et al. 2015) and is required for hyphal polar growth (Li et al. 2020).We further determined that M. oryzae fimbrin could be phosphorylated by Pmk1 at site Ser94 (Figs. 2 and 3).This is a new phosphorylation site for fimbrin.Considering that S94D could rescue the actin organization and hyphal polar growth of Δpmk1 (Figs. 5 and 6), we believe this site is critical for the physiological functions of fimbrin.Furthermore, we found that the mutation of Ser94 significantly decreased actin bundling activity (Fig. 3).In addition, in the Δpmk1, MoFim1 did not localize to the Spk region at the hyphal tip (Fig. 4); thus, we speculate that Ser94 is critical for the Spk distribution of MoFim1 and for protein activity in actin bundling.
Knockout of PMK1 in M. oryzae was first reported to prevent the formation of appressorium and invasive growth in rice plants (Xu and Hamer 1996).Pmk1 was subsequently revealed to be responsible for the control of the hyphal constriction required for fungal growth to penetrate across the plasmodesma, enabling the invasive hyphae to extend from one rice cell to the neighboring cell (Sakulkoo et al. 2018).Recently, it was further revealed that Pmk1 can phosphorylate the Hox7 homeobox transcription factor, which activates a gene expression network to regulate M. oryzae cell division, autophagy, and turgor generation.Pmk1 can also regulate actin cytoskeletal reorganization by phosphorylating the transcriptional regulator Mst12 (Oses-Ruiz et al. 2021).In this study, we found that Pmk1 could also directly interact with the actin-associated protein MoFim1 and regulate its phosphorylation at Ser94 (Figs. 2 and 3).Pmk1 both regulated MoFim1 activity during actin bundling and its localization (Figs. 3 and 4, Supplemental Movies 1 and 2).Like in Δmofim1, actin in the Δpmk1 hyphal tip region was aberrantly organized, and especially at Spk, actin could not be always maintained (Fig. 5B and D, Supplemental moves 4 and 6).Considering that S94D could reverse actin organization at the hyphal tip (Fig. 5E, Supplemental move 7) and hyphal growth defects in Δpmk1 (Fig. 6), we believe that the regulatory effect of Pmk1 on actin organization was associated with MoFim1.Pmk1 is a critical switch in M. oryzae development and infection and can activate a network of genes (Oses-Ruiz et al. 2021).Thus, in our study, MoFim1 alone (and even S94D) could not completely rescue the infection failure of Δpmk1 (Fig. 7), although it partially rescued the morphological defects of Δpmk1 in inducing an appressorium.Overall, our study provides new evidence indicating how Pmk1 regulates actin cytoskeleton organization during M. oryzae development and pathogenesis.

Strains and culture conditions
All M. oryzae isolates used in this study were derived from the sequenced WT strain Y34 (this strain was kindly provided by Prof. LiHuang Zhu, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) (Xue et al. 2012).The wild-type, Δpmk1, Δmofim1 and complemented strains were cultured at 28 °C on complete medium (CM) plates.For conidia production, these strains were maintained in straw rice bran (SRB) medium at 28 °C for 7 days in the dark.

Targeted gene deletion and mutant complementation
The Δpmk1 and Δmofim1 deletion mutants were generated according to previous methods (Liu et al. 2022).Briefly, two approximately 1.5 kb fragments of the sequences flanking PMK1 were amplified with two primer pairs.The obtained DNA fragments were ligated upstream and downstream of the hygromycin gene.Then, the recombinant DNA was infused into the pGKO vector (Zhou et al. 2017) via homologous recombination cloning (ClonExpress MultiS One Step Cloning Kit,Vazyme Biotech,C112).The protoplasts of wild-type Y34 were transformed with pGKO-PMK1 for targeted gene deletion.
To construct plasmids expressing PMK1-mCherry and MoFim1-GFP, approximately 1.5 kb of the native promoter region from the M. oryzae genome was amplified and cloned and inserted into the pKNTG expression vector (Zheng et al. 2016).All the constructs were cloned by homologous recombination (ClonExpress MultiS One Step Cloning Kit, Vazyme Biotech, Nanjing, China; C112); all the primers with restriction enzyme sites are listed in Supplemental Table S1.

Pathogenicity assay
M. oryzae conidia were harvested from 10-day-old SRB agar cultures with sterile water.The samples were filtered through Miracloth and resuspended to a concentration of 5 × 10 4 spores/ml in a 0.2% (w/v) gelatin solution (Li et al. 2017b(Li et al. , 2019)).Then, the conidia were injected into 20-day-old cultured rice (Oryza sativa cv.Nipponbare) sheaths.The infected rice sheaths were maintained under humid conditions at 28 °C.The leaf sheaths were observed under a Zeiss880 confocal microscope.
For infection of the rice leaves, the same region of M. oryzae cultured in SRB media was used to infect punched rice leaves (the third leaf of each seedling, cv.Nipponbare).The inoculated plants were kept in a growth chamber at 28 °C with 90% humidity and a 12 h/12 h light/dark cycle.Lesions from rice leaves were observed after 5 days (Zhang et al. 2011).

Co-IP analysis
To generate the GFP tag constructs, DNA fragments, including GFP MoFim1-GFP, were cloned and inserted into the vector pSulPH (Li et al. 2020(Li et al. , 2017c)).To generate mCherry tag constructs, mCherry and PMK1-mCherry DNA fragments were amplified and inserted into the vector pKNTG.All of the resulting GFP-tag and mCherry fusion constructs were confirmed by sequencing analysis and subsequently transformed into the Y34 strain in pairs.M. oryzae mycelia were cultured in CM liquid medium.The hyphae were ground into powder in liquid nitrogen and resuspended in 1 ml of extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA, and 1 × protease inhibitor cocktail (Sigma, S8830)).After centrifuging for 16,000 g at 4 °C for 30 min, the supernatants were incubated with 10 μL of α-GFP-Trap-fused Magnetic Agarose (ChromoTek, GNA-25-500) for 1 h.The beads were subsequently washed four times with PBS.After the last centrifugation, the PBS was removed completely.Then, 20 μL of SDS-PAGE sample buffer was added, and the beads were boiled for 10 min.The presence of the PMK1-mCherry or MoFim1-GFP was detected by immunoblotting for GFP (Abmart, P60046S) or mCherry (Abmart, P60056S).

Phosphorylated-mass spectrometric analysis
The phosphorylated-mass spectrometric analysis was based on the method described previously (Yan et al. 2018).
Briefly, M. oryzae mycelia in WT or Δpmk1-expressing MoFim1-GFP were cultured in fresh CM liquid medium.The collected MoFim1-GFP M. oryzae mycelia were subsequently ground to powder in liquid nitrogen.The powder was fixed with three volumes of protein extraction buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 10% glycerol; 1 mM DTT; 1 mM EDTA; and 1 × protease inhibitor cocktail (Sigma, S8830)) for 1 h (Gupta et al. 2015).The samples were subsequently centrifuged at 12,000 rpm at 4 °C for 30 min, after which the pellets were discarded.The supernatant was incubated with 20 μL of α-GFP-Trap-fused Magnetic Agarose (ChromoTek, GNA-25-500) for 1 h.The beads were subsequently washed four times with PBS.After the last centrifugation, the PBS was removed completely.Then, 20 μL of SDS-PAGE sample buffer was added, and the beads were boiled for 10 min.The soluble samples were subjected to SDS-PAGE for 10-15 min.Then, the gel was cut (approximately 1 cm) and proteins were excised and collected for digestion with trypsin at 37 °C for 18 h using a trypsin/substrate ratio of 1:50.The resulting peptides were analysed through high-resolution mass spectrometry (SCIEX, USA ZenoTOF 7600).Phosphopeptides were further enriched by IMAC beads (Phos-Select IronAffinity Gel; Sigma-Aldrich).

BiFC experiment
To construct the plasmids for the BiFC experiments, the PMK1 gene with a native promoter was cloned and inserted into the C-terminus of YFP in the PKNTG vector containing a hygromycin resistance gene.The MoFim1 gene with a native promoter was cloned and inserted into the vector PBHT2 containing the bleomycin resistance gene to generate the MoFim1-YFP plasmid.The two plasmids were subsequently transformed into Y34 protoplasts.The obtained M. oryzae clones were examined using fluorescence microscopy (Zeiss880 microscope).All the constructs were generated via homologous recombination cloning (ClonExpress Mul-tiS One Step Cloning Kit; Vazyme Biotech).The primers used are listed in Supplemental Table 1.

Yeast two-hybrid assay
Full-length cDNAs of MoFim1, ABD1, EF and ABD2 were cloned and inserted into the pGADT7 (AD) vector.Full-length cDNAs of the PMK1 gene were inserted into the pGBKT7 (BD) vector.To examine the interactions between Pmk1 and MoFim1, the AD and BD constructs were coexpressed in the yeast strain AH109, and clones were screened on SD-Trp-Leu media.Then, the transformants were isolated and grown on SD-Trp-Leu-His-Ade media.The sequences of primers used are listed in Supplemental Table 1.

In vitro protein purification and F-actin binding/bundling assay
The cDNA fragments containing the MoFim1, S94D, S94A, S117A or PMK1 ORF were infused into the bacterial expression vector pET-28a (Novagen/Merck) with the enzyme sites BamH1 and SalI to produce His-tagged MoFim1s-GFP fusion proteins, including MoFim1, S94A, S94D, S117A and S117D.The constructs were subsequently transformed into Transetta (DE3) chemically competent cells (TransGen, CD801-02).The expression of the fusion proteins was induced by 0.5 mM IPTG for 5 h at 28 °C.These recombinant proteins were purified using ProteinIso Ni-NTA resin (TransGen, DP101-01) according to the manufacturer's instructions.All primers and restriction enzyme sites are listed in Table S1.
In vitro phosphorylation analysis was performed according to previous methods (Feng et al. 2022).The purified Pmk1 alone or in combination with the MoFim1, S94A, or S117A proteins was mixed in kinase reaction buffer (100 mM PBS, 1 mM ascorbic acid (pH 7.5), and 10 mM MgCl 2 with 50 µM ATP at 25°C for 1 h.Then, tenfold cold acetone was added to terminate the reaction.These reaction samples were diluted with 1 × SDSloading buffer (TransGen, DL101-02).The proteins were separated by SDS-PAGE.Then, the phosphorylation of MoFim1 was detected using a commercial antibody that recognizes phosphorylated Ser and Thr (ECM Biosciences, PP2551, 1:1,000 dilution).
Visualization of actin filaments in the presence of the recombinant proteins was performed by fluorescence microscopy as reported previously (Han et al. 2013).Briefly, prepolymerized rabbit F-actin (1 μM) (Cytoskeleton, BK037) was incubated with MoFim1 and its corresponding site mutant proteins (1 μM) at room temperature for 30 min and labelled with Alexa561-phalloidin (Aladdin, P287444).Images were obtained under a confocal microscope (LSM880; Zeiss) at 561 nm.

Observation of fluorescent signals by live-cell imaging and actin analysis
Live-cell imaging experiments were conducted as previously described (Abubakar et al. 2023).Briefly, the mycelia were cultured in liquid CM for 24 h.The mycelia were placed upside down on a glass slide, with the fungal hyphae directly touching the surface of the slide.Then, the growing hyphae were observed under a confocal microscope (LSM880; Zeiss) equipped with an Airyscan detector for time-lapse imaging.The laser lines included GFP (using argon laser excitation at 488 nm and emission spectra at 500-540 nm) and mCherry (using neon laser excitation at 561 nm and emission spectra at 600-640 nm).

Fig. 1
Fig. 1 MoFim1 interacts with Pmk1.A M. oryzae hyphae expressing MoFim1-GFP or GFP were subjected to immunoprecipitation and mass spectrometry (IP-MS).Hyphae of MoFim1-GFP and GFP were collected three times and combined for protein extraction and IP-MS analysis.The most accumulated putative interacting proteins in M. oryzae identified by IP with MoFim1 are listed.B Yeast two-hybrid assay showing the interaction between MoFim1 and Pmk1.Yeast cells containing the indicated plasmids were grown on SD/-Leu/-Trp DO (DDO) plates and SD/-Leu/-Trp/-Ade/-His DO (QDO) plates.AD-T and BD-53 were used as the negative and positive controls, respectively.C Schematic diagram showing the protein domains of MoFim1.D Yeast two-hybrid assay showing the interaction sites between MoFim1 and Pmk1.The indicated domains of MoFim1 were expressed in yeast cells and subsequently grown on SD/ − Leu/ − Trp DO (DDO) plates and SD/ − Leu/ − Trp/ − Ade/ − His DO (QDO) plates.E Co-IP experiment showing the interaction between MoFim1 and Pmk1.M. oryzae expressing the indicated plasmids were used in these experiments.M. oryzae proteins were incubated with anti-GFP agarose beads.Immunoblotting was subsequently conducted with anti-mCherry antibodies.F Bimolecular fluorescence complementation (BiFC) assays showing the interactions between MoFim1 and Pmk1.YFP, (N)-Fim1 and YC-Pmk1 plasmids were constructed and expressed in wild-type M. oryzae.Coexpression of YN and CN was used as a negative control.Bars = 5 μm

Fig. 2
Fig. 2 Pmk1 functions in the phosphorylation of MoFim1.A Pathogenicity assay of the generated Δpmk1, ΔMofim1 and complemented strains after punch inoculation.The same area of each SRB culture plate from the Δpmk1, ΔMofim1 and complemented strains was used to infect these rice leaves (O.sativa cv.Nipponbare).Photographs were taken 5 days after infection.B Quantification of the lesion area on the rice leaves shown in (A).The error bars represent SD (n = 20), and the asterisks (**) represent significant differences (P < 0.01).C and F Phosphorylation analysis of MoFim1 in Δpmk1 hyphae (C) and appressorium (F).Total proteins from Δpmk1 and WT hyphae or 8 h appressorium expressing MoFim1-GFP were extracted and incubated with anti-GFP agarose beads.After washing, the samples were analysed with anti-GFP and anti-phosphorylation antibodies.The number in the figure indicates the fold change in intensity of the band analyzed by ImageJ software.D λ-PP was added to test the phosphorylation of MoFim1.The extracted proteins were incubated with λ-PP before western blotting.The number in the figure indicates the fold change in intensity of the band analyzed by ImageJ software.E Images of appressourim of WT and Δpmk1 used for protein extraction.Bars = 10 µm.G Phosphorylation analysis of MoFim1 in the WT and the Δmofim1

Fig. 4
Fig. 4 Localization and actin bundling analysis of MoFim1.A and B Expression of pMoFim1-MoFim1-GFP in the WT (A) and Δpmk1 (B) strains.The obtained hyphae were observed using high-resolution imaging.The numbers at the top right corner indicate the timestamps (min:s).Bars = 2 μm.C and D Enlarged region at the hyphal tip.E and F Line scan analysis of the discrepancy in the localization of MoFim1-GFP in the hyphal tips of the WT and Δpmk1.G Line scan analysis of the fluorescence in (E) and (F).H Growth rate analysis of the WT and Δpmk1 hyphae according to the observed living hyphae.The data are presented as the mean ± SE; n ≥ 30 cells.Asterisks indicate statistically significant differences according to Student's t test (**p < 0.01)

Fig. 5
Fig. 5 Actin organization in Δpmk1 and ΔMofim1.The hyphae were labelled with Lifeact-GFP to visualize the actin cytoskeleton.Time-lapse images showing actin dynamics in growing hyphae of the WT (A), Δpmk1 (B), complemented strain Δpmk1 (C), Δmofim1 (D) and S94D (E).The red arrows indicate the presence of actin at Spk during polar growth.The numbers at the top right corner indicate the timestamps (min:s).Bars = 2 μm

Fig. 6
Fig. 6 S94D has a function in rescuing the hyphal growth defect of Δpmk1.A Five days cultured M. oryzae on SRB medium including the WT, Δmofim1, the complemented strain of Δmofim1 by expressing ofum MoFim1, S94A, S94D, Δpmk1, Δpmk1 expressing of PMK1, MoFim1, S94A, S94D.B Colony diameter of the indicated M. oryzae strains in (A).The experiments were repeated three times with similar results.The error bars represent SD (n = 20), and the asterisks (**) represent significant differences (p < 0.01)